Project Summary/Abstract Mammalian neurons evolved to have a highly complex architecture that underlies neuronal function. The long-term goal of the proposed work is to understand the elaboration of neuronal architecture on a molecular level. Proper functioning of many neuronal processes is crucially dependent on the correct localization of many proteins at specific cellular locations. Correct localization is ensured by polarized membrane traffic from the trans Golgi network and by endocyctosis and endosomal sorting. My lab studies how neurons accomplish polarized membrane traffic in order to establish distinct domains, such as axons and dendrites. The difference in functions between axons and dendrites requires that many of the molecules underlying signal reception, integration, and propagation be spatially segregated. Various adhesion molecules similarly need to be localized correctly. The axonal cell adhesion molecule L1/NgCAM is crucial to normal brain development; mutations in L1 lead to pleiomorphic neurodevelopmental defects, called CRASH syndrome. In the previous funding period, we developed kinetic, function-interfering, and live-imaging approaches in cultured neurons to determine how the axonal cell adhesion molecule L1/NgCAM travels to the axon. We demonstrated that it does so via a novel route: transcytosis via somatodendritic endosomes. Other cargos, such as transferrin (Tfn) receptors and AMPA receptors, also traverse somatodendritic endosomes, but are not ultimately sorted to axons. The goal of the proposed research is to elucidate the subcellular organization and molecular regulation of polarized membrane transport in neurons, focusing in particular on endosomes. We will determine which compartments and which regulators accomplish the task of sorting different proteins from somatodendritic endosomes to either axons or dendrites. In preliminary work, we already identified three proteins as regulators of NgCAM trafficking, and we will elucidate the roles of NEEP21, syntaxin13 (Aim 1) and Rme1/EHD1 (Aim 2) in the sorting of NgCAM and transferrin. Lastly, we will ask if endosomal trafficking of L1/NgCAM is constitutive or regulated by ligands (Aim 3). The centrality of membrane traffic for neuronal function is underscored by the large number of trafficking regulators genetically linked to neurodegenerative conditions. Uncovering the complexities of neuronal endosomes thus has wide implications for many aspects of neuronal functioning. Probing the role of regulators for L1/NgCAM trafficking in comparison to transferrin will lead to new insights into the neuronal-specific elaboration of a polarized endosomal system. Since L1 expression is upregulated after neuronal injury and after stroke, understanding L1 trafficking to axons has implications for regenerative processes after injury.